CN111875600A - Preparation method of tropane alkaloids in anisodamine - Google Patents
Preparation method of tropane alkaloids in anisodamine Download PDFInfo
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Abstract
The invention discloses a preparation method of tropane alkaloids in anisodamine, which comprises the following steps: pulverizing anisodamine, extracting with methanol acetate solution, filtering the extractive solution with ceramic membrane to remove impurities, separating with organic membrane to remove impurities, and drying the water solution passing through the organic membrane. The preparation method has high extraction efficiency and convenient operation, and is beneficial to industrial production, popularization and application.
Description
Technical Field
The invention particularly relates to a preparation method of tropane alkaloids in anisodamine.
Background
Anisodamine [ Anisodus tanguticus (Maxim.) Pascher ] is perennial herb of the genus anisodamine of the family Solanaceae. The anisodamine is wild in Qinghai, Tibet, Sichuan and Gansu, and grows on hillside and roadside in mountain area with elevation of 2200-. The Tibetan medicine is prepared from anisodamine seeds and roots, and the Tibetan medicine of the roots is named as Tangchonnabao, has the effects of anesthesia, analgesia, disinsection, sedation and detoxification, and is mainly used for treating virus malignant sores, diseases, skin anthracnose, mania and the like. The main effective component of anisodamine is tropane alkaloid such as atropine, scopolamine, anisodamine, etc., and these compounds have the effects of blocking acetylcholine receptor, dilating capillary, improving microcirculation, and resisting shock.
At present, technical reports of extracting tropane alkaloids from plants are few, and in experiments for determining the content of scopolamine and anisodamine in anisodamine, the royal jelly et al adopt alkalescent reagents of ammonia water and chloroform for extraction, so that the method has low extraction efficiency and strong toxicity of chloroform, and therefore, a new extraction method of tropane alkaloids with high efficiency and low toxicity needs to be established.
Disclosure of Invention
In order to solve the problems, the invention provides a preparation method of tropane alkaloids in anisodamine, which comprises the following steps:
pulverizing anisodamine, extracting with methanol acetate solution, filtering the extractive solution with ceramic membrane, and drying the filtrate.
Further, the anisodamine is root and/or aerial part of perennial root herb of the genus Anisodus of the family solanaceae, Anisodus tandutinus (Maxim.) pascher.
Further, the mass-volume ratio of the anisodamine to the methanol acetate solution is 1 g: 5-20 ml, preferably 1 g: 10 ml.
Furthermore, the concentration of acetic acid in the acetic acid methanol solution is 3-5%, preferably 4%.
Further, the extraction is ultrasonic extraction or reflux extraction; the ultrasonic extraction time is 5-60 min, the power is 500-2500w, the preferable time is 30min, and the power is 2500 w; the hot reflux extraction time is 1.5-3h, the temperature is 65-85 ℃, the preferable time is 2.5h, and the temperature is 75 ℃.
Further, the pore size of the ceramic membrane is 0.2 μm or 0.1 μm.
Further, the drying is freeze drying, wherein the parameters of the freeze drying are the temperature of the cold hydrazine is-65 to-40 ℃, and the drying time is 15 to 40 hours.
The invention also provides tropane alkaloids prepared by the method, wherein the total content of the tropane alkaloids is more than 50%.
Further, the tropane alkaloids comprise anisodine, scopolamine, anisodamine and atropine.
The invention finally provides the application of the tropane alkaloid in preparing the anti-acetylcholine receptor medicine.
The preparation method of the tropane alkaloids in anisodamine has high extraction efficiency and convenient operation, is beneficial to industrial production, popularization and application, has high content of the tropane alkaloids prepared by the method, has strong activity, has obvious inhibition effect on smooth muscle contraction as proved by experiments, and has wide prospect in medicines for resisting acetylcholine receptors.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 shows the total ion flow diagram of tropane alkaloids in anisodamine
Detailed Description
Example 1 preparation of tropane alkaloids from anisodamine
Collecting 500g of anisodamine root, pulverizing, adding 5000ml of 4% acetic acid methanol solution, ultrasonic extracting at power of 2500w for 30min, filtering the extractive solution with 0.2 μm ceramic membrane to remove impurities, and freeze drying the filtrate (at-65 deg.C for 15 hr) to obtain tropane alkaloid.
Example 2 preparation of tropane alkaloids from anisodamine
Taking 2kg of anisodamine root, pulverizing, adding 30L of 4% acetic acid methanol solution, ultrasonically extracting for 20min under the condition of power of 1000w, filtering the extract with 0.1 μm ceramic membrane to remove impurities, and freeze-drying the filtrate (the temperature of cold hydrazine is-55 deg.C, and the drying time is 25 hr under the condition of freeze-drying) to obtain tropane alkaloid.
Example 3 preparation of tropane alkaloids from anisodamine
Collecting 4kg of anisodamine root, pulverizing, adding 60L of 4% acetic acid methanol solution, extracting under reflux at 65 deg.C for 1.5 hr, filtering the extractive solution with 0.1 μm ceramic membrane to remove impurities, and freeze drying the filtrate (at-52 deg.C for 30 hr) to obtain tropane alkaloids.
Example 4 preparation of tropane alkaloids from anisodamine
Taking 2kg of anisodamine root, pulverizing, adding 40L of 4% acetic acid methanol solution, extracting under reflux at 55 deg.C for 2 hr, filtering the extractive solution with 0.1 μm ceramic membrane to remove impurities, and freeze drying the filtrate (at-50 deg.C for 25 hr) to obtain tropane alkaloid.
The advantageous effects of the present invention are described below by way of test examples.
Experimental example 1 preparation of tropane alkaloids from anisodamine
Weighing 500g of anisodamine root, crushing, adding 2500ml of 4% acetic acid methanol solution, extracting by three ways of leaching, ultrasonic extraction and reflux extraction respectively, filtering the extracting solution by a 0.2 mu m ceramic membrane to remove impurities, freeze-drying the filtrate for 15 hours at the temperature of minus 65 ℃ of cold hydrazine, and detecting the content of tropane alkaloids in the dried extract by liquid chromatography, wherein the specific extraction conditions and the detection results of the extract are shown in table 1.
The liquid chromatography detection method comprises the following steps: weighing the prepared extract, adding methanol for ultrasonic dissolution, centrifuging the extracting solution, and taking the supernatant for testing; ② accurately weighing anisodine, anisodamine, scopolamine and atropine reference substances respectively, and adding methanol to dissolve to obtain reference substance solution; detecting the sample solution and the reference solution by the following chromatographic conditions: the chromatographic column is Agilent (150mm × 4.6mm i.d.,4 μm); taking 0.1% formic acid solution as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B, and performing linear gradient elution for 0min and 10% B; 20min 10% B; the flow rate is 1 ml/min; the column temperature is 30 ℃; the detection wavelength is 210 nm; sample introduction amount: 10 mu l of the mixture; fourthly, calculating the content of each alkaloid by using an external standard method, wherein the content of the tropane alkaloid is the sum of the content of each alkaloid.
TABLE 1 tropane alkaloid content in extracts obtained by different extraction methods
| Extraction method | Filtration method | Tropane alkaloid content |
| 5% ammonia water chloroform leaching for 24h | Removing impurities from ceramic membrane | 20％ |
| Leaching with 4% acetic acid and methanol for 24 hr | Removing impurities from ceramic membrane | 23％ |
| Ultrasonic extracting with 4% acetic acid and methanol for 0.5h | Removing impurities from ceramic membrane | 58％ |
| Reflux extracting with 4% acetic acid and methanol for 2 hr | Removing impurities from ceramic membrane | 56％ |
As can be seen from table 1, the extraction requires a long time, the obtained extract has a low content of tropane alkaloids, the ultrasonic extraction and reflux extraction not only takes a short time, but also the obtained tropane alkaloids have a content of more than 50%, so the extraction of tropane alkaloids in anisodamine root adopts ultrasonic or reflux extraction.
2. Selection of extraction solvent
Weighing 500g of anisodamine root, crushing, adding 2500ml of acid solutions with different concentrations, performing ultrasonic extraction for 1h at the power of 2000w and performing reflux extraction for 2h at the temperature of 70 ℃, filtering an extracting solution by a 0.2 mu m ceramic membrane to remove impurities, performing freeze drying on a filtrate for 15 h at the temperature of-65 ℃ under the condition of cold hydrazine, and determining the content of tropane alkaloids in the dried extract by using a detection method under the item '1', wherein the specific detection results of an extraction solvent and the extract are shown in Table 2.
TABLE 2 tropane alkaloid content in extracts obtained with different extraction solvents
As can be seen from table 2, in both the reflux extraction and the ultrasonic extraction, the extraction solvent was 3%, the content of tropane alkaloids was more than 50% in the case of 5% methanol acetate solution, and the content of tropane alkaloids was the greatest in the case of 4% methanol acetate solution.
3. Selection of ratio of liquid to feed
Weighing multiple parts of anisodamine root, crushing, adding 4% acetic acid methanol solution with different volumes, performing ultrasonic extraction for 1h at the power of 2000w and reflux extraction for 2h at the temperature of 70 ℃, filtering an extracting solution by a 0.2 mu m ceramic membrane to remove impurities, freeze-drying a filtrate for 15 h at the temperature of-65 ℃ of cold hydrazine, and measuring the content of tropane alkaloids in the dried extract by using a detection method under the item '1', wherein the specific material-liquid ratio and the detection result of the extract are shown in Table 3.
TABLE 3 tropane alkaloid content in extracts obtained by different feed-to-liquid ratios
| Extraction solvent | Purification method | Tropane alkaloid content |
| Ultrasonic extraction with 4% acetic acid and methanol for 1h (ratio of material to liquid 1:5) | Removing impurities from ceramic membrane | 52％ |
| Ultrasonic extraction with 4% acetic acid and methanol for 1h (feed liquid 1:10) | Removing impurities from ceramic membrane | 56％ |
| Ultrasonic extraction with 4% acetic acid and methanol for 1h (feed liquid 1:15) | Removing impurities from ceramic membrane | 54％ |
| Ultrasonic extraction with 4% acetic acid and methanol for 1h (feed liquid 1:20) | Removing impurities from ceramic membrane | 52％ |
| Ultrasonic extraction with 4% acetic acid and methanol for 1h (feed liquid 1:25) | Removing impurities from ceramic membrane | 46％ |
| Ultrasonic extraction with 4% acetic acid and methanol for 1h (feed liquid 1:30) | Removing impurities from ceramic membrane | 42％ |
| Reflux extraction of 4% acetic acid with methanol for 2h (feed liquid 1:5) | Removing impurities from ceramic membrane | 52％ |
| Reflux extraction of 4% acetic acid with methanol for 2h (feed liquid 1:10) | Removing impurities from ceramic membrane | 58％ |
| Reflux extraction of 4% acetic acid with methanol for 2h (feed liquid 1:15) | Removing impurities from ceramic membrane | 57％ |
| Reflux extraction of 4% acetic acid with methanol for 2h (feed liquid 1:20) | Removing impurities from ceramic membrane | 54％ |
| Reflux extraction of 4% acetic acid with methanol for 2h (feed liquid 1:25) | Removing impurities from ceramic membrane | 43％ |
| Reflux extraction of 4% acetic acid with methanol for 2h (feed liquid 1:30) | Removing impurities from ceramic membrane | 42％ |
As can be seen from table 3, under the two extraction modes of reflux extraction and ultrasonic extraction, when the material-liquid ratio is 1: 5-1: 20, the content of the tropane alkaloids is more than 50%, and when the material-liquid ratio is 1:10, the content of the tropane alkaloids is the largest.
4. Extraction time selection
Weighing 500g of anisodamine root, crushing, adding 5000ml of 4% acetic acid methanol solution, performing ultrasonic extraction at the power of 2000w and reflux extraction at the temperature of 70 ℃, filtering an extracting solution by a 0.2 mu m ceramic membrane to remove impurities, freeze-drying a filtrate for 15 hours at the temperature of-65 ℃ of cold hydrazine, and determining the content of tropane alkaloids in the dried extract by using a detection method under the item '1', wherein the specific extraction time and the detection result of the extract are shown in Table 4.
TABLE 4 tropane alkaloid content in extracts obtained at different extraction times
| Extraction time | Purification method | Tropane alkaloid content |
| Ultrasonic extracting with 4% acetic acid and methanol for 10min | Removing impurities from ceramic membrane | <10％ |
| Ultrasonic extracting with 4% acetic acid and methanol for 20min | Removing impurities from ceramic membrane | 52％ |
| Ultrasonic extracting with 4% acetic acid and methanol for 30min | Removing impurities from ceramic membrane | 58％ |
| Ultrasonic extracting with 4% acetic acid and methanol for 40min | Removing impurities from ceramic membrane | 56％ |
| Ultrasonic extracting with 4% acetic acid and methanol for 50min | Removing impurities from ceramic membrane | 55％ |
| Ultrasonic extracting with 4% acetic acid and methanol for 60min | Removing impurities from ceramic membrane | 54％ |
| Ultrasonic extracting with 4% acetic acid and methanol for 90min | Removing impurities from ceramic membrane | 36％ |
| Ultrasonic extracting with 4% acetic acid and methanol for 120min | Removing impurities from ceramic membrane | 34％ |
| Reflux extracting with 4% acetic acid and methanol for 0.5 hr | Removing impurities from ceramic membrane | 29％ |
| Reflux extracting with 4% acetic acid and methanol for 1 hr | Removing impurities from ceramic membrane | 32％ |
| Reflux extraction of 4% acetic acid with methanol for 1.5h | Removing impurities from ceramic membrane | 52％ |
| Reflux extracting with 4% acetic acid and methanol for 2 hr | Removing impurities from ceramic membrane | 54％ |
| Reflux extraction of 4% acetic acid with methanol for 2.5h | Removing impurities from ceramic membrane | 59％ |
| Reflux extracting with 4% acetic acid and methanol for 3 hr | Removing impurities from ceramic membrane | 53％ |
| Reflux extracting with 4% acetic acid and methanol for 3.5 hr | Removing impurities from ceramic membrane | 46％ |
| Reflux extracting with 4% acetic acid and methanol for 4 hr | Removing impurities from ceramic membrane | 42％ |
| Reflux extracting with 4% acetic acid and methanol for 4.5h | Removing impurities from ceramic membrane | 38％ |
| Reflux extracting with 4% acetic acid and methanol for 5 hr | Removing impurities from ceramic membrane | 36％ |
As can be seen from table 4, when the ultrasonic extraction time is 20-60min, the content of tropane alkaloids is greater than 50%, and when the ultrasonic extraction time is 30min, the content of tropane alkaloids is the maximum; when the reflux extraction time is 1.5h-3h, the content of tropane alkaloids is more than 50%, and when the reflux extraction time is 2.5h, the content of tropane alkaloids is the maximum.
5 ultrasonic power optimization
Weighing 500g of anisodamine root, crushing, adding 5000ml of 4% acetic acid methanol solution, performing ultrasonic extraction for 30min under different powers, filtering the extract by a 0.2 mu m ceramic membrane to remove impurities, freeze-drying the filtrate for 15 h at the temperature of-65 ℃ under the condition of cold hydrazine, and measuring the content of tropane alkaloids in the dried extract by using a detection method under the item '1', wherein the specific extraction time and the detection result of the extract are shown in Table 5.
TABLE 5 tropane alkaloid content in extracts obtained by different ultrasonic power extractions
| Extracting power | Purification method | Tropane alkaloid content |
| 4% ultrasonic acetic acid methanol (power 500w) | Removing impurities from ceramic membrane | 53％ |
| 4% ultrasonic acetic acid methanol (power 1000w) | Removing impurities from ceramic membrane | 56％ |
| 4% ultrasonic acetic acid methanol (power 1500w) | Removing impurities from ceramic membrane | 58％ |
| 4% ultrasonic acetic acid methanol (power 2000w) | Removing impurities from ceramic membrane | 63％ |
| 4% ultrasonic acetic acid methanol (power 2500w) | Removing impurities from ceramic membrane | 65％ |
As can be seen from Table 5, the content of tropane alkaloids is more than 50% when the ultrasonic power is 500-.
6 optimization of extraction temperature during reflux extraction
Weighing multiple parts of anisodamine root 500g, crushing, adding 5000ml of 4% acetic acid methanol solution, reflux extracting for 2.5h at different temperatures, filtering the extracting solution by a 0.2 mu m ceramic membrane to remove impurities, freeze-drying the filtrate for 15 h at the temperature of-65 ℃ under the condition of cold hydrazine, and measuring the content of tropane alkaloids in the dried extract by using the detection method under the item '1', wherein the specific extraction time and the detection result of the extract are shown in Table 6.
TABLE 6 tropane alkaloid content in extracts obtained by extraction at different extraction temperatures
| Extracting power | Purification method | Tropane alkaloid content |
| Reflux extraction of 4% acetic acid methanol at 60 deg.C | Removing impurities from ceramic membrane | 53％ |
| Reflux extraction of 4% acetic acid methanol at 65 deg.C | Removing impurities from ceramic membrane | 58％ |
| Reflux extraction of 4% acetic acid methanol at 70 deg.C | Removing impurities from ceramic membrane | 62％ |
| Reflux extraction of 4% acetic acid methanol at 75 deg.C | Removing impurities from ceramic membrane | 66％ |
| Reflux extraction of 4% acetic acid methanol at 80 deg.C | Removing impurities from ceramic membrane | 64％ |
| Reflux extraction of 4% acetic acid methanol at 85 deg.C | Removing impurities from ceramic membrane | 57％ |
| Reflux extraction of 4% acetic acid methanol at 90 deg.C | Removing impurities from ceramic membrane | 48％ |
As can be seen from table 6, the content of tropane alkaloids is greater than 50% at 60-85 ℃ of reflux extraction temperature, and is the greatest at 75 ℃.
7 comparison of purification of different ceramic membranes
Weighing multiple parts of anisodamine root 500g, crushing, adding 5000ml of 4% acetic acid methanol solution, extracting for 2.5h at 75 ℃, filtering by different ceramic membranes to remove impurities, freeze-drying the filtrate for 15 h at-65 ℃ under the condition of cold hydrazine temperature, and measuring the content of tropane alkaloids in the dried extract by using the detection method under the item '1', wherein the specific extraction time and the detection result of the extract are shown in Table 7.
TABLE 7 tropane alkaloid content in extracts obtained after different ceramic membrane purification
| Purification method | Tropane alkaloid content |
| Removing impurities from 0.1 μm ceramic membrane | 63％ |
| Removing impurities from 0.2 μm ceramic membrane | 65％ |
| Removing impurities from 0.5 μm ceramic membrane | 22％ |
As can be seen from Table 7, the content of tropane alkaloids in the ceramic membrane is more than 50% at 0.1 μm and 0.2 μm.
Experimental example 2 Mass Spectrometry characterization of tropane alkaloids in anisodamine
Sample experiment pretreatment method
Weighing 1g of tropane alkaloid prepared in example 1, adding 15ml of methanol, carrying out ultrasonic treatment for 15min, centrifuging at 12000 r/min for 20min, and taking supernatant for testing.
Second, instrument and equipment
UPLC-Triple-TOF/MS system: AcquisytTM ultra high performance liquid chromatograph (Waters, USA), Triple TOF 5600+ type flight time mass spectrometer, equipped with electrospray ion source (AB SCIEX, USA); eppendorf minispan centrifuge (Germany Eppendorf Co.)
Third, test conditions
Chromatographic conditions the column was Watt's acid UPLC HSS T3(150 mm. times.2.1 mm i.d.,1.8 μm); taking 0.1% formic acid solution as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B, and performing linear gradient elution for 0min to obtain 0% B; 2min 0% B; 25min 30% B; 35min 95% B; 37min 95% B; the flow rate is 0.3 ml/min; the column temperature is 50C; the detection wavelength is 254 nm; sample introduction amount: 2 μ l.
Mass spectrometry conditions UPLC-Triple-TOF 5600+ time of flight liquid mass spectrometer: a negative ion scanning mode; scanning range: m/z 100-1500; atomizing gas (GS 1): 55 psi; atomizing gas (GS 2): 55 psi; air curtain gas (CUR): 35 psi; ion source Temperature (TEM): 600 ℃ (positive); ion source voltage (IS): 5500V (positive); primary scanning: declustering voltage (DP): 100V; focus voltage (CE): 10V; secondary scanning: and (3) acquiring mass spectrum data by using TOF MS-Product Ion-IDA modes, wherein CID energy is-20V, -40V and-60V, and before sample injection, performing mass axis correction by using a CDS (compact disc reader) pump to ensure that the error of the mass axis is less than 2 ppm.
Fourthly, analyzing the detection result and the data
The total ion flow diagram of tropane alkaloids in anisodamine is shown in figure 1. The tropane alkaloids of anisodamine comprise anisodamine, scopolamine, anisodamine, atropine, and noratropine. Table 8 shows the secondary mass spectra data for tropane alkaloids.
TABLE 8 Secondary Mass Spectrometry data for tropane alkaloids in anisodamine
Test example 3 content determination of tropane alkaloids in anisodamine according to the present invention
Sample experiment pretreatment method
Weighing 0.5g of tropane alkaloid prepared in example 1, adding 15ml of methanol, carrying out ultrasonic treatment for 15min, centrifuging at 12000 r/min for 20min, and taking supernatant for testing.
Second, instrument and equipment
Agilent 1260 high performance liquid chromatography.
Preparation of control solution
Accurately weighing anisodine, anisodamine, scopolamine, and atropine reference substances respectively at 3.0mg, 2.0mg, and 4.0mg, placing in a 10mL volumetric flask, adding methanol to dissolve, shaking, and fixing to desired volume to obtain anisodine, scopolamine, and atropine reference substance solutions with concentrations of 0.3mg/mL, 0.2mg/mL, and 0.4 mg/mL.
Fourth, test conditions
Chromatographic conditions the chromatographic column was agilent (150mm × 4.6mm i.d.,4 μm); taking 0.1% formic acid solution as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B, and performing linear gradient elution for 0min and 10% B; 20min 10% B; the flow rate is 1 ml/min; the column temperature is 30 ℃; the detection wavelength is 210 nm; sample introduction amount: 10 μ l.
Fifth, the detection result
See Table 9
TABLE 9 tropane alkaloid content prepared in example 1
Test example 4 Choline-inhibiting Activity of the tropane alkaloids of anisodamine of the invention
1. Preparation of rat tracheal smooth muscle specimen
Rats were anesthetized by intraperitoneal injection of 1000mg/kg of 10% chloral hydrate, the chest and neck were cut open, the trachea was rapidly freed, and the rat was rinsed in Krebs-Henseleit (K-H) solution. Carefully remove fat around the trachea and cut the trachea into tracheal rings of about 1cm for use.
2. Rat tracheal smooth muscle tonometry
Rat isolated tracheal smooth muscle specimen is suspended in a bath tank filled with 20mLK-H liquid, a PowerLab data acquisition and analysis system is connected through a tension transducer, the tension is adjusted to about 2.0g, the rat isolated tracheal smooth muscle specimen is balanced at 37 ℃ for 1H, and the nutrient solution is changed for 1 time every 15 min. By 1X 10-5Stimulation of carbachol at mol/LContracting trachea, and adding medicine (i.e. tropane alkaloids prepared in example 1) every 5min by cumulative dose method when the contraction amplitude of trachea is maximum to make the concentration of tropane alkaloids in the nutritional liquid reach 1 × 10-6、1×10-5、2×10-5、1×10-4And (3) mol/L, adding ultrapure water with the same volume into the blank group as a control, and reflecting the change of the tracheal tension by using the ratio of the tracheal tension amplitude after adding the medicine to the maximum carbachol-induced contraction amplitude.
3 results
The results of the inhibition of tropanes in anisodamine are shown in table 10. Compared with the blank control group, the tropanes in anisodamine have inhibiting effect on the contraction of the tracheal smooth muscle of the rat induced by carbachol.
TABLE 10 anticholinergic activity of tropanes in anisodamine
In conclusion, the preparation method of the tropane alkaloids in anisodamine has high extraction efficiency and convenient operation, and is beneficial to industrial production, popularization and application. Experiments prove that the tropane alkaloid prepared by the method has high content and strong activity, has obvious inhibition effect on smooth muscle contraction, and has wide application prospect in medicines for resisting acetylcholine receptors.
Claims (10)
1. A preparation method of tropane alkaloids in anisodamine is characterized in that: it comprises the following steps:
pulverizing anisodamine, extracting with methanol acetate solution, filtering the extractive solution with ceramic membrane, and drying the filtrate.
2. The method of claim 1, wherein: the anisodamine is root and/or aerial part of perennial root herb of Anisodus of the genus anisodamine of the family Solanaceae.
3. The method of claim 1, wherein: the mass volume ratio of the anisodamine to the acetic acid methanol solution is 1 g: 5-20 ml, preferably 1 g: 10 ml.
4. The production method according to claim 1 or 3, characterized in that: the concentration of acetic acid in the acetic acid methanol solution is 3-5%, and the preferable concentration is 4%.
5. The method of claim 1, wherein: the extraction is ultrasonic extraction or reflux extraction; the ultrasonic extraction time is 5-60 min, the power is 500-2500w, the preferable time is 30min, and the power is 2500 w; the hot reflux extraction time is 1.5-3h, the temperature is 65-85 ℃, the preferable time is 2.5h, and the temperature is 75 ℃.
6. The method of claim 1, wherein: the aperture of the ceramic membrane is 0.2 μm or 0.1 μm.
7. The method of claim 1, wherein: the drying is freeze drying, the parameters of the freeze drying are the temperature of the cold hydrazine is-65 to-40 ℃, and the drying time is 15 to 40 hours.
8. A tropane alkaloid prepared according to the method of any one of claims 1 to 7, characterized in that the total content of said tropane alkaloids is more than 50%.
9. The tropane alkaloids of claim 8, wherein the tropane alkaloids comprise anisodine, scopolamine, anisodamine and atropine.
10. Use of a tropane alkaloid according to claim 8 or 9 in the manufacture of a medicament against acetylcholine receptors.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113599439A (en) * | 2021-09-23 | 2021-11-05 | 中国科学院西北高原生物研究所 | Anisodamine alkaloid extraction method based on response surface optimization and preparation thereof |
| CN114414679A (en) * | 2021-12-27 | 2022-04-29 | 吉林省农业科学院 | Method for detecting belladonna alkaloid in animal tissue |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1595059A (en) * | 1976-11-10 | 1981-08-05 | Saint Firmin A R | Tropane alkaloids |
| AU3078489A (en) * | 1988-02-26 | 1989-08-31 | Seitaikinou Riyou Kagakuhin Shinseizougijutsu Kenkyu Kumiai | Method for producing tropane alkaloid |
| CN102850343A (en) * | 2011-06-29 | 2013-01-02 | 中国科学院大连化学物理研究所 | Pretreatment method for alkaloid in Anisodus tanguticus |
| CN110003197A (en) * | 2019-04-23 | 2019-07-12 | 南京久安源环保科技有限公司 | A kind of extraction process of tropane alkaloids |
| CN113599439A (en) * | 2021-09-23 | 2021-11-05 | 中国科学院西北高原生物研究所 | Anisodamine alkaloid extraction method based on response surface optimization and preparation thereof |
-
2020
- 2020-08-10 CN CN202010796863.XA patent/CN111875600A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1595059A (en) * | 1976-11-10 | 1981-08-05 | Saint Firmin A R | Tropane alkaloids |
| AU3078489A (en) * | 1988-02-26 | 1989-08-31 | Seitaikinou Riyou Kagakuhin Shinseizougijutsu Kenkyu Kumiai | Method for producing tropane alkaloid |
| CN102850343A (en) * | 2011-06-29 | 2013-01-02 | 中国科学院大连化学物理研究所 | Pretreatment method for alkaloid in Anisodus tanguticus |
| CN110003197A (en) * | 2019-04-23 | 2019-07-12 | 南京久安源环保科技有限公司 | A kind of extraction process of tropane alkaloids |
| CN113599439A (en) * | 2021-09-23 | 2021-11-05 | 中国科学院西北高原生物研究所 | Anisodamine alkaloid extraction method based on response surface optimization and preparation thereof |
Non-Patent Citations (9)
| Title |
|---|
| 丁琳等: "一测多评法同时测定莨菪浸膏片中3个生物碱的含量", 《药物分析杂志》 * |
| 何丽一等: "中国茄科植物中莨菪烷生物碱的薄层分离和含量测定", 《药学学报》 * |
| 刘劲威等: "消旋山莨菪碱滴眼液含量测定方法研究", 《现代医药卫生》 * |
| 张晓峰等: "山莨菪植物体内4种莨菪烷类生物碱含量的变化", 《西北植物学报》 * |
| 朱若华等: "薄层扫描法测定洋金花中3种莨菪生物碱的研究", 《分析试验室》 * |
| 柯月娇等: "非水毛细管电泳法测定山莨菪中4种莨菪烷类生物碱", 《福州大学学报(自然科学版)》 * |
| 蒋运斌等: "野生山莨菪根的生物碱类成分分析", 《时珍国医国药》 * |
| 许瑛等: "高效液相色谱法测定栽培山茛菪根部莨菪烷类生物碱含量", 《分析试验室》 * |
| 靳宝峰，杨晓军，毕森林，卢建华: "离子对色谱法测定颠茄及莨菪制剂中的3种有效成分", 《药物分析杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113599439A (en) * | 2021-09-23 | 2021-11-05 | 中国科学院西北高原生物研究所 | Anisodamine alkaloid extraction method based on response surface optimization and preparation thereof |
| CN114414679A (en) * | 2021-12-27 | 2022-04-29 | 吉林省农业科学院 | Method for detecting belladonna alkaloid in animal tissue |
| CN114414679B (en) * | 2021-12-27 | 2024-05-07 | 吉林省农业科学院 | Method for detecting belladonna alkaloid in animal tissues |
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